Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

A loxP-transposon retrofitting strategy for generating large nested deletio ns from one end of the insert DNA in bacterial artificial chromosomes and P 1 artificial chromosomes was described recently [Chatterjee, P, K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by usin g primers located in the transposon end to illustrate its value for positio n-specific single-nucleotide polymorphism (SNP) discovery from chosen regio ns of large insert clones. A simple ampicillin sensitivity screen was devel oped to facilitate identification and recovery of deletion clones free of t ransduced transposon plasmid, This directed approach requires minimal DNA s equencing, and no in vitro subclone library generation; positionally orient ed SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned l ibraries or sequence databases. The deletion templates, positioned SNPs, an d markers are also used to orient large insert clones into a contig. The de letion clone can serve as a ready resource for future functional genomic st udies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially a pplicable to the analysis of genomes for which a full genome sequence or ra diation hybrid cell lines are unavailable.